Measurement of glucose in an insulin delivery catheter by minimizing the adverse effects of insulin preservatives

ABSTRACT

In an aspect, the present disclosure provides a combined drug delivery cannula and continuous glucose sensor that measures glucose without interference from the drug excipient, said cannula being a hollow tube, the outer wall of which includes: an electrode layer with at least one indicating electrode, said layer underlying a redox-catalytic layer that includes an osmium compound bound to a ligand, and either glucose oxidase or glucose dehydrogenase.

CROSS-REFERENCE

This application is a continuation of U.S. application Ser. No.16/992,772, filed Aug. 13, 2020, which is a continuation of U.S.application Ser. No. 15/169,432, filed May 31, 2016 (now U.S. Pat. No.10,780,222, issued Sep. 22, 2020), which claims the benefit of U.S.Provisional Application No. 62/170,655, filed Jun. 3, 2015, each ofwhich is incorporated by reference herein in its entirety.

BACKGROUND

People with diabetes are at risk of developing complications such askidney disease, eye disease, cardiovascular disease and foot/nervedisease. It is typically more difficult to control glucose levels inthose who require insulin treatment as compared to those who do not. Allpatients with Type 1 Diabetes (T1D) require insulin and many deliverinsulin by a continuous pump, which allows precise, regulated deliveryof insulin 24 hours per day.

Another valuable technique in managing T1D is Continuous GlucoseMonitoring (GCM), in which a subcutaneously-inserted sensor providesinterstitial glucose data to the user every few minutes. Severalstudies, including the JDRF-sponsored trial, showed that persons of allages who used CGM on a regular basis experienced better glycemic controlthan non-users, as measured by hemoglobin A1C (A1C) (JDRF CGM StudyGroup. JDRF randomized clinical trial to assess the efficacy ofreal-time continuous glucose monitoring in the management of type 1diabetes: research design and methods. Diabetes Technol Ther. 2008;10(4):310-21). However, this and other studies found that many patientsfound CGM usage cumbersome and many used CGM only sporadically. Notsurprisingly, when used sporadically or rarely, CGM usage did not leadto a better glycemic control in the JDRF trial.

Daily life can be difficult for those who regularly use both an insulinpump and CGM. Such individuals must indwell two through-the-skindevices, which can increase the risk of pain, infection and other sideeffects compared to a single device. Persons with T1D typically carrymultiple devices on the body, for example, a pump and/or syringes, a CGMreceiver, a vial of insulin, a blood glucose monitor for calibrating theCGM, and blood glucose monitoring strips and lancets. The multiplicityof devices leads to a situation known as “device burden,” which can leadto frustration, anger and often causes a patient to choose among devicesrather than utilizing all the devices that can improve his/her health.

Because of these issues regarding device burden, there is an unmet needto integrate a CGM and insulin pump cannula into a single device.

Manufacturers' instructions state that a subcutaneous glucose sensormust be located far away from the insulin pump cannula site. In supportof this statement, during pig studies, we found that current insulinformulations markedly interfere with currently-available hydrogenperoxide-measuring sensors. More specifically, we found that thepreservatives in the formulations, such as phenol and m-cresol, areelectroactive and interfere with CGM.

In the current invention, we teach a method in which the sensor can besuccessfully integrated with the insulin cannula.

DESCRIPTION OF THE RELATED ART

Rather than using a separate insulin infusion catheter and a CGM sensor,it is desirable to create a single combined device. There are manydifferent strategies for glucose sensing that could be considered forsuch a combined sensing catheter. For example, there is prior artregarding the use of optical sensing technologies for glucose.US20130040404A1 to Crane et al teaches an optical glucose sensor builtupon an optical waveguide. US20050118726 A1 to Schultz et al teaches anoptical sensing method based upon a glucose-binding fusion protein.US20130060106 to Aasmul et al teaches an optical fiber-based sensorhaving a hollow fiber filled with a glucose binding assay. WO 2000064492A1 by Schultz and Ballerstadt et al teach a porous hollow sensorcontaining porous beads for the optical determination of analyteconcentration. Alternative sensing strategies such as viscometry havealso been disclosed (eg U.S. Pat. No. 6,210,326 B1 to Ehwarld). However,none of these is well-suited to pair CGM with drug infusion in a singledevice.

A common analyte sensor design is based upon the principle ofamperometry, in which analytes are detected by generation of anelectrochemical signal related to the analyte of interest. The sensingelectrodes are commonly fabricated through the use of sputtered orevaporated thin films deposited on the surface of a substrate. Often,indicating electrodes (also known as working electrodes) are made ofplatinum, gold or carbon. When a positively biased indicating electrodeis coupled with a reference electrode, such as silver/silver chloride,redox-active analytes can be amperometrically detected. With theaddition of an enzyme layer such as glucose oxidase, a sensor can bemade quite specific for the analyte glucose. The glucose oxidase is ableto convert glucose, which is not readily detected amperometrically, intohydrogen peroxide which is readily detected. When thin films of metalelectrodes are deposited on an appropriate polymer film such aspolyimide, the resulting sensor has the added advantage of flexibility.Users might find a rigid catheter or needle uncomfortable or painful.

One problem with electrodes made from metallic thin films is fragility;the layers can delaminate when exposed to physical trauma such asimpact, flexion, shear stresses, and tensile stresses. For example,Alzoubi et al found that durability of thin film electrodes is limited.More specifically, a large number of flexion cycles led to materialsfailure, a phenomenon known as cycle fatigue (Alzoubi K, Lu S, SammakiaB. Experimental and Analytical Studies on the High Cycle Fatigue of ThinFilm Metal on PET Substrate for Flexible Electronics Applications. IEEETransactions, Manufacturing. 2011; 1:43-51). While the durability of athin film may be sufficient for short-term applications, longer termambulatory sensing applications require a much greater ability towithstand trauma. In the case of indwelling subcutaneous sensors, thesensor must withstand repeated impacts and/or repeated flexion over aperiod of time lasting from 3 to 7 days or beyond. Alzoubi found thatthin metal films underwent cracking which was aggravated by immersion inwet, high-salt environments such as those presented by mammalian bloodor subcutaneous interstitial fluid. Consequently, the electrodes in theleading commercially-available CGM sensor (made by Dexcom, Inc) areconstructed from durable solid wires rather than thin films. Examples ofthis design can be found in many patent disclosures. U.S. Pat. No.8,812,072 B2 to Brister et al teaches a wire-based variable stiffnesstranscutaneous medical device. U.S. Pat. No. 8,543,184 B2 to Boock et alteaches a wire-based transcutaneous implantable continuous analytesensor with a silicone-based membrane. U.S. Pat. No. 8,060,174 B2 toSimpson et al teaches a biointerface for a wire-based sensing electrode.U.S. Pat. No. 8,515,519 B2 to Brister et al teaches a transcutaneousanalyte sensor assembly. U.S. Pat. No. 5,165,407 to Wilson et al teachesa flexible, solid wire-based glucose sensor. U.S. Pat. No. 7,471,972 B2to Rhodes et al teaches a multi-electrode wire-based sensor. U.S. Pat.No. 9,131,885 B2 to Simpson et al teaches a multi-layer sensor having asolid core. However, a wire or rod has a solid core and is thus notcompatible with delivery of a drug such as insulin, which requires ahollow lumen. None of these devices would be suitable for combinedanalyte sensing and drug delivery due to their lack of a hollow lumen.

Earlier inventions have disclosed sensors coupled with hollow catheters.In U.S. Pat. No. 8,886,273 to Li, Kamath, and Yang, the inventors teacha glucose sensor disposed within a hollow catheter. More specifically,the sensor in this invention is disposed inside a larger diametercatheter that is indwelled inside a blood vessel. Whereas such aninvention is appropriate for measuring a liquid (blood) that existswithin a catheter, such a design is not appropriate for a sensingcatheter which is intended for measuring glucose in subcutaneous fattytissue. For use in subcutaneous tissue, the sensing elements must be onthe outer wall of the hollow catheter. Stated differently, a “wiresensor within a tube” or “tube within a tube” design will not allowproper function in subcutaneous tissue. For drug delivery, the innerlumen must be hollow. Similarly, in U.S. Pat. No. 6,695,958 B1 to Adamet al, the authors disclose a device having sensing elements located inthe interior of the hollow part and designed to measure analytes in theinterior lumen. For an effective subcutaneous sensing catheter, it isnecessary to have an open interior (lumen) to allow for drug deliveryinto the body. In an embodiment of our invention, the outer wall, whichis not in contact with a drug and which is bathed withglucose-containing subcutaneous interstitial fluid, is the optimallocation for the sensing elements.

Other sensor configurations require the withdrawal of fluid samples fromthe body in order for sensing to occur. U.S. Pat. No. 5,174,291 A toSchoonen et al discloses a hollow fiber-based glucose sensor thatinvolves dialysis with a test solution. CA 2347378 A1 to Knoll et alincorporates a hollow probe for the withdrawal of interstitial fluid. EP1327881 A1 to Beck at al teaches a hollow electrochemical cell withinternal sensing elements requiring the drawing up of the fluid sample.U.S. Pat. No. 8,277,636 B2 to Sode et al teaches a glucosedehydrogenase-based sensor incorporating an interstitial fluid samplingdevice. US20060000710 A1 to Weidenhaupt et al teaches a method fordetermining glucose concentration that requires the use of a device thathas an external sensor coupled with a fluid-sampling pump. US20110180405A1 to Chinnayelka teaches a sensor incorporating a hollow member and alancet for the sampling of interstitial fluid. U.S. Pat. No. 5,176,632 Ato Bernardi teaches a system that incorporates a microdialysis-basedsensor. U.S. Pat. No. 6,605,048 B1 to Levin et al teaches a samplingdevice that incorporates a vacuum for the drawing up of a blood samplefrom the skin surface. None of these devices would permit ongoingdelivery of a drug with simultaneous exposure of the sensor tointerstitial fluid. Consequently, these systems are not compatible withcontinuous subcutaneous drug infusion.

Other sensor configurations utilize microneedles to reduce theinvasiveness of the measurement technique, such as the invention that isthe subject of 20060025717 to Zimmerman et al. However, the chiefproblem with microneedle arrays is the difficulty of keeping all themicroneedles indwelled in mammalian tissue during body movement. Becausemicroneedles are short in length, many of the needles will have atendency to come out of tissue when the person moves suddenly orforcefully. This problem of unintentional explantation renders themunsuitable for extended use in an outpatient setting.

An application by Yang et al (US20160136357) discloses a unified hollowstructure that can be used for analyte sensing and for drug delivery.Though insulin is specifically mentioned, this invention does notinclude any means of avoiding the oxidative interference frompreservatives and does not include a method of avoiding the fragility ofthin metal electrodes laminated to hollow structures. Similarly, apatent application US 20150374905 to Yodfat et al does not enablemeasurement of glucose in the presence of insulin preservatives and doesnot avoid the problem of fragility of thin metal electrodes.

In order to fabricate a combined sensor/catheter, one can incorporatebiosensing elements into the wall of a hollow needle or catheter. Themost obvious and simplest strategy would be to directly deposit metal(e.g. platinum, gold) indicating thin film indicating electrodes andthin film silver (Ag/AgCl) reference electrodes on the underlyingpolymer layer such as polyimide or polyester. One such design, disclosedin WO2002039086 to Ramey et al, incorporates printed electrode films.However, after carrying out many studies in animals, we have observed amajor problem with sensing catheters made of thin film metal electrodesdeposited over a polymeric layer. These sensors exhibited frequentdelamination and general lack of durability.

SUMMARY

At many bias potentials, insulin preservatives (phenol and m-cresol) inthe vicinity of a glucose sensing indicating electrode create a largecurrent (flow of electrons) which is not readily distinguishable from ahigh glucose level. More specifically, when an indicating electrode inthe presence of the preservatives is polarized at a high bias potential,there is a large current even in the absence of glucose. For thisreason, one method of reducing or eliminating the glucose-like currentis to use a much lower bias potential. If a hydrogen peroxide sensingsystem is utilized, it is difficult to achieve a sufficient glucosecurrent from peroxide oxidation while, at the same time, minimizing theinterference that results from the insulin preservatives.

In contrast, if one utilizes certain systems such as osmium-based redoxmediators that operate at a low bias potential, electrons can betransferred from glucose to an indicating electrode without interferencefrom the insulin preservatives. In such a case, the mediator can be heldin place by attachment to a polymer such as polyvinylpyridine orpolyvinylimidazole which can be further crosslinked by bifunctionalcrosslinkers and immobilized at the sensor surface.

In addition to, or instead of, using redox mediator chemistry, aspecialized filter can be used to trap the phenol and m-cresol beforebeing delivered to the patient, thus preventing these compounds fromreaching the subcutaneous space and causing an interference current.Because these filters prevent the phenol and m-cresol from reaching thesubcutaneous space and from reaching the amperometric sensor, suchfilters can be used in combinations with sensors that employconventional hydrogen peroxide detection, such as platinum-based sensorswithout redox mediators.

Regardless of whether a redox mediator or filter is utilized, the devicewill not function properly if the layers of the sensing catheter are notdurable. For example, if thin metal films that make up the indicatingelectrode are deposited directly on to the polymer substrate, theelectrode films will not be robust and durable. Instead, they willdisintegrate and/or delaminate from the polymer during the usage period.

To avoid this fragility, and at the same time, minimize cost, it isnecessary to laminate the thin metal electrode films to an underlyingmetal such as titanium. To be sufficiently robust, this metal must besubstantially thicker than the electrode film.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1, a graph, shows the current obtained from a platinum electrodepolarized at 600 mV. The initial response to hydrogen peroxide is normaland stable. The subsequent responses to ascending amounts of an insulinformulation containing phenolic preservatives initially display apositive (oxidative) response and later show a continuous decline ofcurrent, typical of electrode poisoning.

FIG. 2, a bar graph, shows the electrochemical effects of phenol andm-cresol on bare electrodes polarized at many different bias potentials.In particular, at high positive potentials typical of those used forperoxide detection, a very high oxidative current is observed. For bothphenol and cresol, the oxidative current declines markedly as the biaspotential is lowered. The appearance of such data obtained with platinumand gold electrodes is very similar. The data shown here are on goldelectrodes.

FIG. 3 shows the structural formula of the polymer repeat unit(poly(l-vinyl imidazole)) 1 bound to osmium 4 with two 4,4′-dimethyl,2,2′-bipyridine moieties 2 and 3 (abbreviated PVI-OsDiMeBPY) in additionto one chloride remaining from the original osmium salt ligands.

FIG. 4, a graph, compares the incremental change in current density oftwo types of glucose oxidase-based sensors after exposure to high dosemixed phenolics (total concentration 180 μg/ml, composed of equal partsby weight of phenol and m-cresol) over 20 minutes in the presence ofglucose 5 mM. Over this period, there was a marked decline in currentdensity 5 in each of the three platinum sensors. The bias potential ofthese sensors was 600 mV. In contrast, there was very little change ineach of the three gold sensors 6 that were coated with glucose oxidaseand PVI-OsDiMeBPY and biased at 180 mV.

FIG. 5, a graph, shows a series of amperometric responses of a goldsensor coated with glucose oxidase and PVI-OsDiMeBPY, crosslinked withpolyethylene glycol diglycidyl ether, to successive increases in glucoseconcentration in a solution of phosphate buffer sparged with Argon. Theresponses to glucose over this concentration range are largely linear.

FIG. 6, a graph, shows the amperometric signal 7 (small closed symbols)and glucose levels 8 (large open symbols) obtained in pigs from aglucose oxidase-based hydrogen platinum sensor biased at 600 mV. Whenlis-pro insulin was given at minute 105, the preservatives in thisformulation led to an immediate very high oxidative response followed byelectrode poisoning. The poisoning is evident toward the end of theexperiment when the amperometric signal is minimal despite very highglucose levels.

FIG. 7, a graph, shows the amperometric signal 9 and glucose levels 10obtained in pigs from a gold-based sensor coated with glucose oxidaseand PVI-OsDiMeBPY, crosslinked with glutaraldehyde, biased at 180 mV.Lis-pro insulin was given at minute 105, and despite a high level ofpreservatives in this formulation, there was no change in theamperometric signal 9. Notable is the brisk rise of the current 9 overthe final hour of the experiment in response to marked hyperglycemia,verifying absence of electrode poisoning.

FIG. 8 is a drawing of an in-line filter that removes phenolics from aninsulin infusion line. The proximal feed insulin line 11 brings insulinformulation from an insulin pump and is connected to the proximal end ofthe filter 12. A protective membrane 13 prevents the filter materialfrom entering the insulin exit line 14 located at the distal end of thefilter. A zoomed-in view on the right panel also shows a cylindricalretainer 15 which holds the protective membrane 13 and the exit line 14firmly on to the distal end of the filter 12 without blocking thepassage of insulin.

FIG. 9, a graph, demonstrates the effectiveness of the filter thatremoves phenolics. Aspart insulin was placed on the proximal end of thefilter. Many samples (each 0.25 ml in volume) were then collected duringdelivery of phosphate buffer (in 0.25 ml units). Results of an assay forinsulin 16 (as estimated by measurement of total protein using abicinchoninic acid [BCA] assay) are quantified on the left axis andresults of an assay for phenol 17 (using a nitroprusside-based assay)are quantified on the right axis. The filter material is Sephadex G10(medium). Insulin passes through the filter very soon after delivery andthe phenolics come out very late, only after 3 ml has passed through thefilter.

FIG. 10 shows the steps in which microfabrication is used to create thepatterns for the electrodes and the interconnect traces. After titaniumfoil is laminated to polyimide, a layer of silver is sputtered over thetitanium, followed by placement of a layer of photoresist. Some of thephotoresist is selectively removed 18 in order to make unwanted silveravailable for removal by an etchant. After the remainder of thephotoresist is removed, the silver electrode pattern is revealed 19.After the next coat of photoresist is applied, platinum is thendeposited by sputtering 20. As the photoresist is removed, the unwantedplatinum is lifted off, revealing the platinum electrode pattern 21. Thenext layer of photoresist is applied and removed selectively 22. Wherephotoresist is absent, it is possible to etch away unwanted titanium. Asthe photoresist is removed 23, the correct titanium pattern is revealed23.

DETAILED DESCRIPTION

To reduce device burden, it is desirable to be able to measure glucosecontinuously at the direct site of insulin delivery, especially insubcutaneous interstitial fluid. In an attempt to better understand theresponse of an electrode or sensor in the presence of an insulinformulation, the experiment shown in FIG. 1 was carried out. This figureshows the responses of a platinum electrode (polarized at 600 mV vs aAg/AgCl reference electrode) studied in phosphate-buffered saline (PBS).(For the purposes of definition, the term reference electrode in thisdisclosure refers to a reference electrode in a three electrode systemor to a combined reference plus counter or reference plus auxiliaryelectrode in a two electrode system). The electrodes were bare, i.e. notcoated with enzyme or an outer membrane. Early in the experiment,hydrogen peroxide (H₂O₂) was added and the electrodes responded brisklyand maintained current in a stable fashion. At minute 13, a standardcommercially-available insulin formulation (aspart insulin, NovoNordisk) was added such that the concentration of phenol and m-cresoltogether (phenolics) was equal to 45 μg/ml. It can be clearly seen thatthere was a brisk oxidative (rising) current immediately after theinsulin formulation was given. However, the rise in current wastransient and after a few minutes, it began to decline despite continuedpresence of the phenolics. At minute 23 and minute 33, more aspartinsulin was given such that the concentration of phenolics became muchhigher, as indicated. It is important to note that no rise in currentwas seen after these later additions; instead, the current continues todecline such that the final current was markedly lower than the originalcurrent obtained from the H₂O₂ (which also remained in the solution).This progressive loss of current is due to poisoning of the electrode.More specifically, the phenol and cresol undergo a process ofelectropolymerization in which a thin layer of insulating polymer isformed on the electrode surface. This layer is largely impermeable tomultiple analytes including H₂O₂, and for this reason, after exposure tophenolics, such an electrode is useless for the purpose of measuringglucose or other analytes.

Another experiment (not shown) was carried out with insulin that doesnot contain preservatives (this preparation was branded Gibco and waspurchased from Thermo Fisher Life Technologies). This insulin did notcause any electrochemical response and did not cause electrodepoisoning. This experiment demonstrates that the interference noted inFIG. 1 is due to the preservatives, not the insulin per se.

We then decided to investigate the effect of the magnitude of the biaspotential on the electrochemical response to the phenol and cresol, asshown in FIG. 2. In this experiment, bare gold electrodes were polarizedat different potentials and exposed sequentially to pheno, 10 mM and tom-creso, 10 mM. Electrodes were exposed to the phenolics for a veryshort period of time and were cleaned between tests to remove anyelectropolymerized polymer. The results showed that the responses to thephenolics are very dependent upon the magnitude of the bias potential.In particular, as the potential is raised to high potentials such asthose over 350 mV, there is a very large oxidative response. Incontrast, as the bias is lowered, particularly below 250, the responseis quite low.

In an attempt to minimize the interference by reducing the biaspotential, we carried out experiments using an osmium redox-mediatedchemistry scheme very similar to that described in the early 1990's bythe team of Heller and Ohara (Ohara T J, Rajagopalan R, Heller A.“Wired” enzyme electrodes for amperometric determination of glucose orlactate in the presence of interfering substances. Anal Chem. 1994;66(15):2451-7).

Osmium complexes are suitable compounds for accepting electrons fromglucose oxidase, more specifically from the prosthetic group of glucoseoxidase known as flavin adenine dinucleotide (FAD). In one embodiment,the osmium is coordinated to a ligand such as, 4,4′-dimethyl2,2′-bipyridine and also bound to the PVI polymer. Many other ligandscan be used. The bond to the PVI prevents the osmium ligand fromdissociating from the polymer backbone. Those skilled in the art willunderstand that electron donating groups such as methyl, methoxy oramino, when bound to the pyridine or imidazole ligands, will allow theosmium to transfer electrons at a lower polarizing bias. The term forthe osmium and the pyridine or imidazole ligand complex is redoxmediator. For optimal function, the redox mediator is bound to a polymerand this complex is known as the redox mediator polymer (RMP).Optimally, the RMP is crosslinked with agents such as glutaraldehyde orpolyethyleneglycol diglycidyl ether, both of which link amine groups.

In one embodiment of the invention, the RMP is deposited on a goldindicating electrode, but other materials may be used, such as vitreouscarbon, glassy carbon, graphite, platinum, or iridium. It is alsopossible to make the indicating electrode porous, for example by the useof acid anodization, laser poration, or plasma etching.

In one embodiment, the RMP is coated with a polymeric layer called theouter membrane. Oxygen permeability is not necessary for the function ofthis type of sensor, but a degree of glucose permeability is necessary.The outer membrane can be made of polyurethane, Nafion,poly(vinylpyridine), poly(vinylpyridine)-co-styrene, molecular weightcutoff polymeric membranes, silicone, hydrogels and many other materialsthat allow glucose permeation.

For the exemplary experiments shown here, we utilized the osmiumcompound shown in FIG. 3. The polymer backbone 1 is composed ofpoly(l-vinyl imidazole) (PVI). Two coordination ligands,4,4′-dimethyl,2,2′-bipyridine 2 and 3 are bound to osmium 4. The osmium4 is bound to approximately one of every 5 to 15 imidazole groups on thePVI.

Using RMP-based sensors and conventional platinum H₂O₂-sensing sensors,we then carried out the in vitro experiment shown in FIG. 4. In thisexperiment, gold sensors with coats of RMP, glucose oxidase, and anouter membrane were compared to platinum sensors coated with glucoseoxidase and an outer membrane. The RMP-based sensors were biased at 180mV and the platinum sensors were biased at 600 mV. After first beingexposed to a 5 mM glucose solution, the platinum-based sensors 5 and theRMP-based sensors 6 were then exposed to ascending amounts of commercialaspart insulin formulation (Novo Nordisk) containing phenolics, asdescribed for the earlier experiments shown in FIG. 1. FIG. 4 shows onlythe incremental response obtained at the highest concentration ofphenolics, 180 μg/ml. Each bar represents results from a single sensor.It should be noted that there was a large negative response in theconventional platinum-based sensors 5 and only a small positive responsein the RMP-based gold sensors. As discussed earlier, this large negativeresponse lowered the response of the sensors to a point far below theoriginal response to glucose (which remained in the solution), thusdemonstrating a degree of electrode poisoning. As a follow up (notshown) to this experiment, both the platinum-based and RMP-based goldsensors were removed from the solution, rinsed and re-exposed to glucose5 mM. The platinum-based sensors had a very low, nearly absent responseto glucose (verifying permanent poisoning), but the response of theRMP-based sensors was brisk and nearly identical to the originalresponse to glucose.

FIG. 5 shows a response of a RMP-based sensor with a gold indicatingelectrode and a Ag/AgCl reference electrode to stairstep increases inglucose concentration in phosphate buffer during continuous Argonsparging. In this case, the RMP and glucose oxidase were crosslinkedwith polyethyleneglycol glycidyl ether but similar results were alsoobtained using glutaraldehyde liquid or glutaraldehyde vapor. Note thatthe response to glucose, up to at least 25 mM, is essentially linear.

The foregoing series of experiments demonstrate that a gold sensor,coated with RMP and crosslinked glucose oxidase and polarized at 180 mVvs Ag/AgCl, is able to measure glucose with little or no interferencefrom the preservatives used in insulin formulations. In contrast, theuse of a platinum sensor, coated with crosslinked glucose oxidase andpolarized at 600 mV, undergoes an initial very large oxidation currentwhen exposed to phenolics. Furthermore, if such exposure lasts for morethan a few minutes, the electrode is consistently poisoned by a denselayer of electropolymerized phenolic compound that prevents H₂O₂ andother common analytes from reaching the indicating electrode and beingmeasured.

Subcutaneous devices are exposed to many types of trauma, for exampledue to bodily motions and impacts typical of daily life. Therefore, eventhough the chemical layers over an electrode can successfully measureglucose without interference from phenol and cresol, a dual use sensingcatheter will not function accurately for its entire usage period unlesssaid catheter has a durable, robust construction.

One method of creating a continuous sensor built into the wall of aninsulin infusion cannula is to laminate flexible thin metal films on theouter wall of a hollow tubular structure. However, if the choice ofmaterials and processes are not carried out correctly, the resultingelectrode layers will be very fragile. More specifically, if thin filmmetal electrode materials (less than 100 nm in thickness) are placeddirectly over polymeric surfaces (with or without underlying thinadhesion layers such at Ti, Au or Ni) the device becomes fragile. Theelectrode films often delaminate or disintegrate during impact, andtherefore, such a device is not adequate for use as a catheter indwelledfor days in the subcutaneous space. In fact, in such a design,substantial electrode delamination can be seen after only a few hours ofin vivo use. In the experience of the inventors, whether or not a 25-200nm tie (adhesion) layer is deposited under the electrodes, such a designleads to a frequent separation of the tie layer from the polyimide,frequent separation of the indicating or reference electrode films fromthe tie layer, and frequent fragmentation of the metal layers.

On the other hand, if a metallic foil is placed beneath the thin filmmetal electrodes, durability and fatigue resistance are markedlyimproved, while maintaining sufficient flexibility for fabrication anduse as a biosensor. The use of the term “foil” indicates a metal layerthat is at least 2 micrometers (μm) in thickness, that is, much thickerthan the thin film layer typically deposited by sputtering, evaporation,printing or electroplating. Discussions of the beneficial mechanicalproperties of foils can be found in three scientific articles (Alzoubi,see above citation; Matsui Y, Ando N, Yoshida T, Kurotobi R, MatsushitaT, Ohno K. Modeling high adsorption capacity and kinetics of organicmacromolecules on super-powdered activated carbon. Water Res. 2011;45(4):1720-8; and Lavvafi H, Lewandowski J R, Lewandowski J J. Flexbending fatigue testing of wires, foils, and ribbons. Materials Scienceand Engineering. 2014; 601:123-30). For these reasons, a metal foil(underneath the thin electrode film) is well-suited for the purpose ofdurability.

All layers of the sensing catheter must be tightly adhered to theadjoining layers. One method of creating interfaces with good adhesionand good durability is the use a laminating press at high temperatureand high pressure. A high tack adhesive such as B-stage acrylate islocated at the interface of the foil and underlying polymer and adheresthe two materials together. After the lamination, thin film electrodematerials can be deposited over the durable metal foil. The thickness ofthe metal foil is typically 2-15 μm.

The metal of which the foil is composed must be chosen carefully. In thecase of an amperometric glucose sensor, the indicating electrode istypically platinum, gold or carbon. Copper (which is commonly used asthe foil for flexible electronic circuits), is not suitable for use in abiosensor. Specifically, if there is concurrent physical contact betweeninterstitial fluid, copper and platinum, a large galvanic current willoccur as a result of the junction of dissimilar metals. A suitablecandidate for the foil is titanium, which is inexpensive and which wefound to cause little to no galvanic current when paired with platinum.Silver and copper are not suitable as this foil material. Gold is ofintermediate value.

Using the durable sensing catheter design discussed above, we carriedout a series of studies in non-diabetic Yucatan mini-pigs of weight33-60 kg. In preparation for this study, sensing catheters werefabricated. A polyimide strip, 12.5 μm in thickness, was laminated witha sheet of titanium foil, 5 μm in thickness. Three thin film 1 sq mmplatinum indicating electrodes and a Ag/AgCl reference electrode weresputtered on to the titanium foil surface. This electrode strip waswrapped around, and laminated to, the outer surface of a blunt, hollow21 gauge stainless steel tube with the aid of an epoxy adhesive designedfor use in high salt moist environments. The indicating electrodes werecoated with glucose oxidase and bovine serum albumin (BSA) (in a ratioof 3:2) and crosslinked with glutaraldehyde vapor, then coated with asilicone-polyurethane copolymer outer membrane (Lubrizol, Inc). The termused for such a dual use device is a “glucose sensing catheter” or“sensing catheter.” A series of interconnect traces emanate from thethree indicating electrodes and the single reference electrode andterminate in a body-worn electronic sensor module which is in electricalcontinuity with the sensing catheter. The sensor module contains abattery and a Bluetooth-enabled transceiver, which transmits theelectrochemical signals to a personal computer or mobile phone.

Multiple sensing catheters, attached to telemetric sensor modules, wereinserted in the subcutaneous tissue of the pig's abdomen underisoflurane anesthesia. The sensor module was adhered to the skin withcyanoacrylate glue, then each pig was allowed to recover fromanesthesia. The next morning, the animal was again anesthetized withisoflurane. After a stabilization period, a euglycemic clamp was carriedout for 5 hours. More specifically, an infusion of 20% dextrose wasgiven intravenously according to a computerized algorithm in order toavoid hypoglycemia. At minute 105 during the clamp, as indicated by thearrow in FIG. 6, lis-pro insulin (0.22 units per kg total dose, dividedbetween two catheters, so that 0.11 units per kg was delivered througheach catheter) was given through some sensing catheters. Insulin was notdelivered through other sensing catheters.

FIG. 6 shows mean exemplary data obtained from several sensing cathetersthrough which the lis-pro insulin was delivered. The electrochemicalsensor current 7 and the blood glucose values 8 (measured in duplicateby a Bayer Contour Next meter) are shown by arrows. Note thatimmediately after the insulin was given, there was a very large currentspike with a fast exponential decline. Late in the experiment, at minute300, a rapid infusion of 20% dextrose was given intravenously, leadingto a marked rise in blood glucose to a level of almost 300 mg/dl. It canbe seen that the sensors were unable to respond vigorously to thismarked rise in glucose level. There was only a very small rise incurrent during hyperglycemia, typical of sensors that had undergoneelectrode poisoning. Many such experiments were carried out in pigs. Insummary, in about 40% of the experiments in which lis-pro insulin wasgiven, there was a marked oxidative rise in current between minutes 105to 165, despite the fact that glucose was being held steady. It islikely that in these cases, the insulin formulation, after leaving thecatheter, flows back on to the sensor elements, causing an oxidativesignal. In the other cases, it is likely that the insulin formulationflows away from the catheter without contacting the sensing elements,thus failing to cause an interfering signal.

Other pig experiments were carried out with sensing catheters with goldindicating electrodes and RMP (PVI osmium 4,4′-dimethyl 2,2′-bipyridine)bound to glucose oxidase by glutaraldehyde. FIG. 7 shows mean data fromseveral RMP-based gold sensors through which lis-pro insulin wasdelivered. Consistent with the in vitro data discussed above, there waslittle to no evidence of interference from the insulin preservativesafter the insulin formulation was given. Sensor current 9 did not riseat minute 105 when lis-pro insulin was given. Furthermore, the RMP-basedsensors responded vigorously to the marked hyperglycemia during the lasthour of the study. Note that blood glucose rose briskly during the lasthour of the study. During this rise, the brisk rise in current 9verified absence electrode poisoning.

We also discovered another means of avoiding the preservative-inducedoxidative current: the use of filter that is placed in the insulininfusion line. U.S. Pat. No. 5,936,061 to Andersson et al, taught theuse of hydrophobic Zeolite filtration to remove insulin preservativesfrom an insulin formulation vial prior to injection. In an embodiment ofthe current invention, we teach the use of in-line filters designed tobe used by persons with diabetes who use portable insulin pumps thatdeliver insulin subcutaneously. Such a filter is depicted in FIG. 8. Theplastic tubing that comes from the insulin pump 11 is attached to thefilter cartridge 12 that is filled with filter material. At the distalportion of the filter cartridge is a protective membrane 13 thatprevents filter beads or particles from being released into the insulintubing (and thus into the body of a patient). One such embodiment forthis protective membrane is porous cellulose acetate, the pore sizebeing smaller than the filter bead material. Many other membranecompositions and many pore sizes are suitable in the fabrication of theprotective membrane. Tubing 14 brings the filtered insulin out of thefilter cartridge into the sensing catheter. On the right panel of FIG. 8is shown a zoomed-in figure of the filter with additional detail.Typically, it is necessary to utilize a retainer unit 15 that holds thefilter cartridge 12, the protective membrane 13 and the exit tubing 14firmly in place. In some embodiments it is also desirable to place aretainer unit at the proximal end of the filter.

There are many such bead or particle materials that can be used tofilter the phenolics out of the insulin formulation. Some of thesematerials include those typically used for size exclusionchromatography, also known as gel filtration chromatography andmolecular sieve chromatography. For the purposes of this invention, sizeexclusion media are defined as particles containing pores that trapsmaller molecules and allow larger molecules to readily pass through. Inthe case of one embodiment of this invention, preservatives contained ininsulin formulations, including m-cresol and phenol, are trapped withinsmall pores. The larger insulin molecules, which are not trapped, passreadily through the filter.

One suitable filter material is crosslinked dextran, one brand of whichis Sephadex®. Sephadex G10 is suitable since it is intended to separatecompounds smaller than 700 Daltons from those larger than 700 Daltons.This grade of crosslinked dextran is suitable because cresol and phenolweigh about 100 Daltons, whereas insulin and insulin analogs weigh about5800 Daltons. Other grades of crosslinked dextran can also be used. Inaddition to dextran, other choices for filter materials include carbon(including charcoal and activated carbon), alumina, silicates, silica,mixtures of alumina and silica knows as Zeolites, and other compoundsused to separate compounds based on molecular size. It is also possibleto use materials typically used in reversed phase high performanceliquid chromatography to separate molecules on the basis ofhydrophobicity/hydrophilicity. Phenol and cresol are more hydrophobicthan insulin.

FIG. 9 shows results of an experiment carried out in order to separateaspart insulin from its preservatives. A filter similar to that shown inFIG. 8 was fabricated using Sephadex medium G10 beads, 40-120 μm (GEHealthcare, Inc). Ten units of aspart insulin were placed on the column,which was 3 mm in diameter and 64 mm in length. Subsequently, 0.25 ml ofPBS was delivered by an insulin pump every 5 minutes and an equal volumeof eluent was collected every 5 minutes. The eluent was assayedrepeatedly for insulin by using a BCA total protein assay (trace 16 inthe figure). Phenol was assayed repeatedly using a nitroprusside-basedassay with a spectrophotometric end point (trace17). The results showthat the insulin elutes very early in the experiment with little to nomore insulin coming out after the second collection. In contrast, phenoldoes not elute until late in the experiment after 3 ml have beencollected. These results demonstrate that this embodiment of theinvention will work well for persons with diabetes who use an insulinpump. The insulin reservoirs for currently-available pumps contain up to3 ml of insulin formulation. Thus, for pump users who use such a filter,the phenolics will not appear during the usage period of 3 days duringwhich no more than 3 ml of insulin formulation can be administered.

A variation on the use of a filter material is to electrically connectto the filter material and remove interfering substanceselectrochemically. As an example, activated carbon filtration particlespacked and immersed in a saline solution are conductive; thus the carboncan be used as an indicating electrode, polarized at 400-800 mV by apower source vs a suitable reference electrode such as a Ag/AgClelectrode. In such a case, to avoid a short circuit, the referenceelectrode cannot be touching the carbon and so a sheath can surround thereference electrode. The sheath prevents contact with the carbon and thesaline allows electron flow to complete the anode-cathode circuit. Thecarbon, when suitably biased, oxidizes and electropolymerizes the phenoland m-cresol rather than allowing their passage through the filter. Insuch a filter, some of the phenolics may adsorb normally on to thecarbon, while at the same time, other phenolics are electropolymerizedto a thin layer of plastic which stays on the carbon in the disposablefilter. The use of electropolymerization and adsorption is moreefficient than adsorption alone.

It is important to note that there are many physical forms that thefilter could take other than the single long tubular structure shown innFIG. 8. For example, it is possible for the filter to loop back onitself many times in a serpentine fashion. Such a design would not takeup as much longitudinal distance.

For use in a person with diabetes, the filter can be placed anywhere inthe insulin delivery line, such as in the insulin reservoir (which isusually situated with the pump body), the insulin tubing, or the insulinfluid path within the skin worn sensor module immediately proximal tothe entry of the fluid into the sensing catheter.

When a filter is used, it is possible to use a higher polarizingpotential bias in order that the sensing system can utilize standardsensing of hydrogen peroxide. In such a case, there is no need for aredox mediator. To optimize the signal from hydrogen peroxide oxidation,a high bias such as over 500 mV is typically used. Alternatively, onecan use the filter in combination with a redox mediated system with alower bias. Such a combination has the advantage of using two effectivemethods in order to markedly reduce the adverse effects of phenol andcresol during CGM.

The above description teaches the use of a filter used to removephenolics from an insulin delivery line after the insulin formulation isplaced in the pump reservoir but before insulin is pumped into a dualuse sensing catheter. However, it should also be noted that such afilter can be used in a standard insulin infusion set (without a glucosesensor). It is important to note that there are many toxic effects ofphenol and m-cresol. These compounds have been associated with cancer,especially bladder cancer {Garrett, 1975 #3}. The US EnvironmentalProtection Agency cites associations between phenolics administrationand weight loss and neurotoxicity {(IRIS), #2; Agency, 2002 #12} and hasclassified m-cresol as category C (possible human carcinogen). Inaddition, these compounds have been associated with many other adverseeffects including inflammation at the site of insulin delivery {vanFaassen, 1989 #10;van Faassen, 1990 #9}). More recently, phenolics haveclearly been shown to be cytotoxic to mammalian cells {Weber, 2015 #17}.

For these reasons, many people who take insulin may decide that they donot want to be exposed to the high concentrations of phenolics that arein all formulations of insulin intended for human use. Therefore, evenin the absence of a sensing catheter, the insulin infusion set withphenolics filter is a useful invention applicable to those persons whouse an infusion pump. For those who do not use an insulin pump, it ispossible to use the same filtration materials to remove phenolics fromcommercial insulin formulations before administering the drug byinjection.

EXAMPLE 1: Redox Mediator-Based Sensing Catheter

LAMINATE METAL FOIL TO POLYMER SUBSTRATE:

Purpose: This step creates a laminate of titanium and polyimide (Ti/Pi).In this example, the Ti thickness is 5 μm and the polyimide thickness is12.5 μm, though these dimensions should not be construed as limiting.This example creates a laminate rectangle whose dimensions are 60 mm×85mm.

Materials include Deionized water; Polyimide sheet w/B-stage acrylateadhesive; Titanium foil; press pads; Teflon sheets, and graphite pressplates, Heated hydraulic press capable of achieving 400 deg F.;

Plate setup process: Between the caul (pressure-applying) plates of thehydraulic press, materials should be stacked in the following order,from bottom to top: Graphite press plate; press pad; Titanium foil;Polyimide, with B-stage adhesive facing titanium foil; press pad;Graphite press plate.

Prepare graphite plate, graphite foil, and Teflon sheets prior tohandling polyimide and titanium. All sheets should be cut to the size ofthe caul plates and cleaned with isopropyl alcohol (IPA), followed bycareful inspection for lint or contaminants.

For operation of the press: Place plate stack into hydraulic press andapply 5000 lb of force to caul plates. Set temperature setting to 375deg F. for both top and bottom plates. Once both caul plates reach 375deg F., set press to 15000 lb and leave in place for 1 hour. Allow caulplates to cool to under 100 F, then remove plate stack from press.

General Equipment and Supplies (for all following steps): Double-sidedpolyimide tape; plastic card; razor blade; 50×75 mm glass slide;isopropyl alcohol (IPA); deionized (DI) water; Pt (platinum) target; Ag(silver) target; aluminum foil; Ar (argon) plasma etcher; quartz crystalmicrobalance (QCM); sputter tool; hot plate; mask aligner-e.g OAI 200tabletop mask aligner; spin coater capable of 300 RPM; argon source.

PREPARE TI/PI LAMINATE FOR APPLICATION OF GOLD AND AG ELECTRODES

Clean glass slide using soap and tap water, IPA wash, DI rinse, Arplasma clean for 1 minute; dry. Place double sided polyimide tape on hotplate. Apply polyimide tape, remove bubbles. Place aluminum foil on hotplate; apply double sided polyimide on slide and place rigid backeradhesive side up. Apply Ti foil to rigid backer. Apply Ti/polyimide plusrigid backer to the polyimide tape. The stack order should be (bottom totop): glass slide, double sided polyimide tape, rigid backer,Ti/polyimide laminate with Ti side up.

DEPOSITION OF SILVER FILM

Purpose: To deposit a layer of Ag (later chloridized to Ag/AgCl) inorder to create reference electrode. The nominal thickness is 400 nm inorder to allow for a reasonable thickness of Ag/AgCl afterchloridization (chloridization reduces the thickness of Ag). In thisprocess, silver sputtering is used, but other methods such as thermalevaporation, printing, or electroplating can also be used. Materialsrequired include: Treated 50×75 mm Ti/PI sheet on glass slide, sputterunit such as CRC-100, Ag target, and Ar compressed gas.

In order to sputter Ag layer, the substrate is placed in the sputterunit, and the vacuum pumps degas any exposed adhesive. The sputterchamber is filled with Ar, the operator allows system to equilibrate to7 mTorr. Sputter until Quartz Crystal Microbalance (QCM) reading is 5.00kA (500 nm) of Ag. (Gain=75, Density=10.5, Z-ratio=0.529, ToolingFactor=256). Remove device from sputter unit. Tape test in a corner with3M Magic Scotch tape to ensure good adhesion. Store in a dust-freecovered container.

AG PATTERNING AND ETCH (REMOVE UNWANTED AG).

For drawings of the main microfabrication (electrode patterning) steps,see FIG. 10. Purpose—To pattern photoresist for Ag pads on Ti/PIsubstrate. Materials: 50×75 mm Silver sputtered Ti/PI substrate on glassslide; NaOH pellets; 300 mL beaker; 250 mL beaker; optical mask, S1813(photoresist); 80/20 primer (80% Propylene Glycol Monomethyl EtherAcetate and 20% Hexamethyldisilazane (HMDS) primer). Materials for cleanroom use include the Mask aligner; Spinner; hotplate; DI water; scale;S1800 series photoresist; NaOH (pellets or solution).

First, carry out the general photoresist process that is included below.Then mix Ag etch solution. Add 75 mL of 3% USP grade H₂O₂, then 8 mLlaboratory grade 30% ammonium hydroxide to a crystallizing dish. Immersepatterned substrate in solution for 30 seconds, gently agitating.Bubbles will not form when the reaction is complete. Rinse with DI waterand blow dry with nitrogen gas or Argon. Remove photoresist with 0.3MNaOH solution.

AU PATTERNING, SPUTTERING, AND LIFTOFF

Purpose: To pattern Au pads on Ti/PI/Ag substrate. Materials include:50×75 mm Silver sputtered Ti/PI substrate on glass slide; NaOH pellets;300 mL beaker; 250 mL beaker; optical mask; S1813 primer; Ti/PI/glasswith Ag deposited on surface; 80/20 primer, as detailed above; Ag etchfilm mask; 3 mL pipette; Acetone; isopropyl alcohol (IPA); crystallizingdishes; graduated cylinder; timer.

Carry out general photoresist process that is included below. Cleanunder Ar for 1 minute. Activate vacuum system. Sputter 90 nm (0.900 KA)Pt. Sputter 50-90 nm of Au (Density=19.3, Z-ratio=0.381). Use Scotchtape to entirely cover the substrate. Press down firmly across thesubstrate, then slowly remove in order to remove Au layer. Inspect tapetest sheet for any failures in Au adhesion. Use an additional piece oftape to remove any bridges between Au pads. Remove photoresist/remainingPt/Au by tape method (3m magic tape over entire array), then sonicate in0.5M NaOH. If any bridges remain, gently scrub using Kimwipe while insolution.

TITANIUM ETCH (REMOVE UNWANTED TI IN ORDER TO CREATE ELECTRICALINTERCONNECTS)

Purpose: To define and separate titanium traces on sensor. It isimportant to prevent titanium that underlies an indicating electrode oran indicating electrode interconnect trace from contacting titanium thatunderlies other indicating electrodes/traces or contacting titanium thatunderlies reference electrodes/traces. Materials include Ti/Pi mountedslide; titanium etchant; 400 mL beaker; crystallization dish; DI water;NaOH, Ultrasonic cleaner.

Carry out general photoresist process that is included below. Prepareetchant bath. Place substrate in etchant solution and observe closely,rinse with DI water when etch is complete.

Rinse with DI water and blow dry with nitrogen gas or argon.

PREPARE SENSORS FOR HUMAN USE: INDIVIDUALIZE, WRAP, CHLORIDIZE, APPLYPROTECTIVE COAT TO REFERENCE ELECTRODE, AND CLEAN INDICATING ELECTRODES

Individualize each tri-electrode strip using mechanical or photonicmeans such as UV laser (wavelength: 405 nm).

Wrap the electrode strip around a 21-25 gauge stainless steel needle(sharp bevel on end) or blunt tube. Electrode strips are wrapped axiallyaround the needle/tube and adhered using epoxy or other biocompatibleadhesive. If a blunt tube is used, a sharpened stylet within the tube isutilized in order to pierce the skin upon insertion. (The stylet islater removed, allowing drug delivery via the lumen of the tube).

Ferric chloridize with 50 mM FeCl₃ for 5-10 min. ALTERNATIVE:Electrochloridize at 0.6 V×10 min using power supply configures so thatthe Ag is the Anode (+) and Pt is the cathode (−). Bath forelectrochloridization is KCl and HCl, both 0.5 M.

Voltage cycle (clean) indicating electrodes in 1×PBS, −1.5 volts×5 min,1.5 volts×5 min, −1.5 volts×5 min. Verify presence of evolving bubblesat sites of electrodes.

Application of redox mediator polymer and glucose oxidase to surface ofgold indicating electrode. In this example, the redox mediator polymerlisted is Poly-(1-vinyl)-imidazole-Osmium-4,4′-dimethyl-2,2′-bipyridine. However, there are suchcompounds that can be used, either with pyridine or imidazole-basedosmium ligands and with poly vinylpyridine, poly vinylimidazole or otherpolymers as the backbone.

Before beginning this step, gold tri-electrodes have been wrapped,cleaned, and chloridized.

Using DIW as a solvent, prepare 1 ml of both of the following solution:redox mediator polymer (10 mg/ml) and glucose oxidase, 100 units per mg(10 mg/ml). Combine 40 uL of the redox mediator solution and 10 uLglucose oxidase solution. When dispensed manually, one can draw up thismixture into 1 mL plastic syringe with 30 gauge needle and carefullyposition tip of needle over center of each of the three electrodes, thendispense a small drop (1 ul) on to each electrode without coating thereference electrode. After partial drying, one can apply a second layerof the mixture. Alternatively, one can use a microdispensing unit suchas ink jet printer, being careful not to heat the enzyme to over 50 degC.

Place the holder upright within a glutaraldehyde vapor chamber (25%glutaraldehyde) for 30 minutes, then let cure for 30 min at roomtemperature.

The outer membrane deposited over the entire shaft including indicatingelectrodes and reference electrode can be one of many glucose permeablepolymers, including polyurethane, silicone, combinedsilicone-polyurethane, or other polymer. One effective outer membrane isPoly-(4-vinyl or 2-vinyl) pyridine co-styrene (10-30% styrene, PVP-S) 64mg/ml, in anhydrous ethanol. One can deposit this polymer manually, byusing a automated dip-coater, using an ink jet printer, micro-contactprinting, or by using other precise method of dispensing. Coat the outermembrane material on the entire sensor shaft. Dry for 15 min at roomtemp.

After drying, it is possible to test the sensor in solutions of glucose,interfering compounds, etc.

ASSEMBLE into ELECTRONIC MODULE that serves the purposes of TELEMETRYand application of POLARIZING BIAS:

Insert the sensing catheter into a battery powered telemetry module(such as a low energy Bluetooth module such as that marketed by Nordic,Inc).

For the redox mediator polymer approach discussed above, a potentialbias of 180 mV is suitable. A low bias such as this largely avoids thesignal artifact resulting from oxidation of insulin preservatives(phenol, m-cresol) that would be seen if a higher bias were used. A lowbias also avoids the problem of electropolymerization that is routinelyseen with the use of higher bias potentials. When larger bias potentialsare used, the cresol and/or phenol undergo the process ofelectropolymerization which deposits a cohesive thin layer of insulatingplastic on the electrode. This layer of plastic reduces or eliminatesthe ability of osmium from communicating with the electrode materialsand also reduces transport of molecules such as hydrogen peroxide to thesurface of the indicating electrode.

STERILIZATION:

Expose to e-beam, gamma irradiation, ethylene oxide or activatedglutaraldehyde sterilizing solution.

ATTACH TO INSULIN PUMP and OPERATE DEVICE:

After priming with insulin, an infusion line from an insulin pump (e.g.Medtronic Minimed, Animas Ping, Tandem t-slim, Roche Spirit, etc) isattached to the sensing catheter (which is located in subcutaneoustissue) and insulin is delivered. The constant pressure head from thefluid infusion line prevents fluids from coming back out of the body. Inorder to be displayed to the user, the glucose concentration or theelectrical current or voltage data representing glucose concentration isobtained from the sensor. These data are transmitted by Bluetooth orother wireless protocol to the display of the insulin pump, to acomputer, to a dedicated medical device, or to a cell phone. Storage ofdata can be carried out on any of these devices or on the body wornelectronics unit that directly interfaces with the subcutaneous sensingcatheter. An advantage of storing the glucose data on the body-worn unitis that the data are not lost if the receiving unit is lost or out ofrange.

GENERAL PHOTORESIST PROCESS (COMMON TO MULTIPLE STEPS).

Materials: 50×75 mm Ti/PI substrate on glass slide; NaOH pellets orsolution; 300 mL beaker; 250 mL beaker; optical mask; photoresist. 80/20primer as defined above.

Method: Mix 200 ml 0.1M NaOH (8 g/L w/pellets or 15 mL/L w/10M solution)primary developer in glass dish. Ensure that solution is well mixed,especially if using NaOH pellets. Mix 0.075 M NaOH secondary rinse inglass dish. Ensure that solution is well mixed. Spincoat 3 mL 80/20primer with standard method-10 seconds @ 1000 RM followed by 30 seconds@ 3000 RPM. Bake for 3 minutes @ 85 C. Spin coat three layers ofphotoresist with standard method. Bake substrate for 1 minute at 85 Cbetween each spin step. Expose for 180 s @ 600W. Bake for an additional60 seconds. Develop in 0.1M NaOH developer, gently agitating. Rinse insecondary bath for 10 seconds. Dry with nitrogen gas, inspect fordeveloped regions with remaining resist. (Exposed regions should appearuniform across the entirety of the substrate. Properly cleaned regionswill gain a faintly white appearance as they go from wet to try if nophotoresist remains on the surface). Bake for 10 minutes and allow tocool. If regions remain, immerse in primary and secondary baths for anadditional 5 seconds and check again. If substantial regions remain, airdry, clean with 0.3M NaOH, and return to step 4. Check processparameters.

EXAMPLE 2: Filtration Using a Platinum Indicating Electrode with HighBias Potential

Many aspects of this example are the same as in Example 1. However,instead of Au being deposited, Pt is deposited by sputtering, usingthese sputter settings: Density=10.5, and Z-ratio=0.529.

No redox mediator is used. Glucose oxidase is applied along with aprotein extender, bovine serum albumin. Glutaraldehyde crosslinker isused to link the amine groups of glucose oxidase and albumin and theweight ratio of glucose oxidase:albumin:liquid glutaraldehyde is betweenranging from 6:4:5 and 6:4:1. The mixture applied to the Pt electrode isdried for at least 10 min at 40° C. Additional layers can be depositedto increase sensitivity to glucose. In such a case, dry the final coatfor at least 20 min. Then rinse in stirred DIW for 10-15 minutes toremove unbound enzyme. Deposit two coats of outer membrane composed of1.5-2.5% w/v polyurethane (PU) or copolymer of silicone and polyurethanedeposited on the indicating electrode(s) and reference electrode(s).Vendors such as AdvanSource Biomaterials, Lubrizol, or DSM Polymers makesuch polymers. The proportion of silicone is used to regulate oxygenpermeation; and polyethylene oxide or poly ethylene glycol moieties orother polar moiety is used to regulate glucose permeation. A suitablesolvent is a mixture of THF and DMAC (25:75, VN). Dry each PU coat×20min at 40 deg C. Keep solvent and polymer/solvent dry with molecularsieves 3A or 4A.

A suitable material for the filter is Sephadex G10, which is rated toseparate compounds with molecular weights above 700 Da from those below700 Da. A suitable tubular structure of approximately 3 mm in internaldiameter and at least 64 mm internal length is filled with the Sephadexmedium G10 beads, size 40-120 μm. The filter is placed in the fluid pathof the insulin formulation. The distal end of the filter is surroundedwith a porous cellulose acetate membrane for the purpose of preventingthe Sephadex gel from entering the fluid path and being delivered to thepatient. The pore diameter of the cellulose acetate is 0.22 μm. Beforeadding the insulin formulation to the filter, the filter beads areideally exposed to an aqueous buffer such as phosphate buffer in orderto swell the beads.

1.-34. (canceled)
 35. A method for delivering an insulin or insulinanalog formulation and measuring subcutaneous glucose concentration,comprising: (a) obtaining a device comprising: a hollow tube comprisinga proximal end and a distal end, wherein the proximal end is in fluidcommunication with a source of the insulin or insulin analogformulation; and an amperometric glucose sensor located no more than 7millimeters (mm) away from the distal end, wherein the amperometricglucose sensor comprises: an electrode layer comprising at least oneindicating electrode, wherein the electrode layer underlies aredox-catalytic layer comprising (1) an osmium-based redox mediatorcomprising an osmium compound covalently bound to a pyridine-based orimidazole-based ligand, and (2) an enzyme comprising glucose oxidase orglucose dehydrogenase; (b) delivering the insulin or insulin analogformulation to the subject subcutaneously via the distal end of thehollow tube, wherein the insulin or insulin analog formulation comprisesan excipient comprising a phenol or cresol; and (c) measuring thesubcutaneous glucose concentration of the subject, at least in part byusing the osmium-based redox mediator and the enzyme to allow electrontransfer from subcutaneous glucose to the at least one indicatingelectrode sufficient to cause a response of the amperometric glucosesensor to the subcutaneous glucose concentration at an applied biaspotential of no more than +250 millivolts (mV) relative to a referenceelectrode, wherein the applied bias potential of no more than +250 mVrelative to the reference electrode allows the electrode layer toundergo substantially no electropolymerization of the excipient duringcontinuous operation of at least one hour of the amperometric glucosesensor, thereby maintaining a sensitivity of the amperometric glucosesensor to the subcutaneous glucose concentration in presence of theinsulin or insulin analog formulation.
 36. The method of claim 35,wherein the device further comprises a housing comprising an upperaccessible surface and a lower surface, and wherein the method furthercomprises adhering the lower surface to a skin surface.
 37. The methodof claim 35, wherein the amperometric glucose sensor is disposed on asecond hollow tube comprising a second distal end, wherein the methodfurther comprises inserting the second distal end subcutaneously. 38.The method of claim 35, wherein the at least one indicating electrodecomprises gold, carbon, graphite, platinum, or iridium.
 39. The methodof claim 35, wherein the ligand is pyridine-based.
 40. The method ofclaim 39, wherein the ligand is 4,4′-dimethyl-2,2′-bipyridine.
 41. Themethod of claim 35, wherein the ligand is imidazole-based.
 42. Themethod of claim 35, wherein the osmium-based redox mediator is bound topoly (4- vinyl pyridine).
 43. The method of claim 35, wherein theosmium-based redox mediator is bound to poly (1-vinyl imidazole). 44.The method of claim 35, wherein the excipient comprises the phenol. 45.The method of claim 35, wherein the excipient comprises the cresol. 46.The method of claim 35, wherein the amperometric sensor is located nomore than 5 mm away from the distal end of the hollow tube.
 47. Themethod of claim 35, wherein the amperometric sensor is located no morethan 3 mm away from the distal end of the hollow tube.
 48. The method ofclaim 35, wherein the reference electrode comprises a silver/silverchloride (Ag/AgCl) reference electrode.
 49. The method of claim 35,wherein the amperometric sensor further comprises an insulating layerand a metal layer, wherein the insulating layer is coupled to the metallayer, and wherein the metal layer is coupled to the electrode layer.50. The method of claim 49, wherein the insulating layer comprises apolyimide.
 51. The method of claim 49, wherein the metal layer has athickness of at least 2 micrometers (μm).
 52. The method of claim 49,wherein the metal layer comprises titanium, gold, or platinum.
 53. Themethod of claim 35, wherein the electrode layer comprises a film havinga thickness of less than 500 nanometers (nm).